Defining single molecular forces required to activate integrin and notch signaling

Abstract

Cell-cell and cell-matrix mechanical interactions through membrane receptors direct a wide range of cellular functions and orchestrate the development of multicellular organisms. To define the single molecular forces required to activate signaling through a ligand-receptor bond, we developed the tension gauge tether (TGT) approach in which the ligand is immobilized to a surface through a rupturable tether before receptor engagement. TGT serves as an autonomous gauge to restrict the receptor-ligand tension. Using a range of tethers with tunable tension tolerances, we show that cells apply a universal peak tension of about 40 piconewtons (pN) to single integrin-ligand bonds during initial adhesion. We find that less than 12 pN is required to activate Notch receptors. TGT can also provide a defined molecular mechanical cue to regulate cellular functions.

Fig. 1

Principle of Tension Gauge Tether (TGT). A ligand for a membrane receptor is immobilized on the surface through a tether which ruptures if the tension applied by the cell through the receptor is larger than its tension tolerance T_tol. Signaling through the receptor is not activated if the tension required for activation exceeds T_tol and ruptures the tether (A) and is activated if T_tol is larger than the required tension (B). (C, D, E). DNA duplex helix as a tether with tunable T_tol values.

Fig. 2

Cell adhesion on TGT with RGDfK. (A) Phase contrast images of adherent CHO-K1 cells (white dots) on circular regions coated with nine TGT constructs of RGDfK-conjugated dsDNA with the indicated T_tol values. (B) Zoomed in phase contrast images of CHO-K1 cells on four of the regions. (C). Cell density (counts per 0.05 mm²) as a function of T_tol for five different cell lines as indicated. (D) Cell density vs. T_tol for CHO-K1 cells in hypertonic vs. isotonic medium. (E) Cell density vs. T_tol for CHO-K1 cells with and without 50 μM Blebbistatin. All error bars denote the standard deviation (n=8).

Fig. 3

Confocal fluorescent images of CHO-K1 cells. (A) 43 pN TGT surface. (B) 56 pN TGT surface. Actin in green and vinculin in red. Images were obtained after 2-hour cell plating. Bright green lines in (B) are stress fibers that terminate in focal adhesion complexes marked in red.

Fig. 4

H2B-YFP expression in the nucleus as a reporter of Notch activation. (A–D) YFP fluorescence images of CHO-K1 cells show nuclear fluorescence of H2B-YFP when Notch is activated. Histograms of fluorescence intensities of single cells are also shown. (a.u.: arbitrary unit) (A) T_tol=12 pN. (B) T_tol=58 pN. (C) 12 pN TGT with DNase I digestion. (D) A control with unconjugated BSA. Here, we used BSA for TGT immobilization and surface passivation because PEG surface did not survive two days period required for our Notch reporter system.